42 VIRAL IMMUNOLOGY Volume 19, Number 1, 2006 © Mary Ann Liebert, Inc. Pp. 42–53 Production of Rotavirus-Like Particles in Tomato (Lycopersicon esculentum L.) Fruit by Expression of Capsid Proteins VP2 and VP6 and Immunological Studies SERGIO SALDAÑA, 1 FERNANDO ESQUIVEL GUADARRAMA, 2 TERESA DE JESÚS OLIVERA FLORES, 3 NANCY ARIAS, 2 SUSANA LÓPEZ, 4 CARLOS ARIAS, 4 ROBERTO RUIZ-MEDRANO, 5 HUGH MASON, 6 TSAFRIR MOR, 6 LIZ RICHTER, 6,7 CHARLES J. ARNTZEN, 6 and MIGUEL A. GÓMEZ LIM 1 ABSTRACT A number of different antigens have been successfully expressed in transgenic plants, and some are currently being evaluated as orally delivered vaccines. Here we report the successful expression of rotavirus capsid proteins VP2 and VP6 in fruits of transgenic tomato plants. By western blot analy- sis, using specific antibodies, we determined that the VP2 and VP6 produced in plants have mole- cular weights similar to those found in native rotavirus. The plant-synthesized VP6 protein retained the capacity to form trimers. We were able to recover rotavirus virus-like particles from tomato fruit (i.e., tomatoes) by centrifugation on a sucrose cushion and to visualize them by electron mi- croscopy. This result indicated that VP2/VP6 can self-assemble into virus-like particles (VLPs) in plant cells, even though only a small proportion of VP2/VP6 assembled into VLPs. To investigate immunogenicity, adult mice were immunized intraperitoneally (i.p.) three times with a protein ex- tract from a transgenic tomatoes in adjuvant. We found that the transgenic tomato extract induced detectable levels of anti-rotavirus antibodies in serum; however, we did not determine the contri- bution of either the free rotavirus proteins or the VLPs to the induction of the antibody response. These results suggest the potential of plant-based rotavirus VLPs for the development of a vaccine against rotavirus infection. 1 Departamento de Ingeniería Genética, Centro de Investigación y de Estudios Avanzados del IPN, Irapuato, México. 2 Facultad de Medicina, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, México. 3 Facultad de Química, Paseo de la Investigación Científica, Universidad Nacional Autónoma de México, México. 4 Departamento de Genetica del Desarrollo y Fisiología Molecular, Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico, Cuernavaca, Morelos, México. 5 Departamento de Biotecnología y Bioingeniería, CINVESTAV-IPN, México, DF, México. 6 Biodesign Institute and School of Life Sciences, Arizona State University, Tempe, Arizona. 7 Present address: Lexicon Genetics Inc., The Woodlands, Texas. INTRODUCTION R OTAVIRUS BELONGS to the Reoviridae family, and is an important cause of neonatal diarrhea in human beings and other animal species. Rotavirus infections are also the most important cause of severe infantile gas- troenteritis, accounting for more than 125 million cases of diarrhea and an estimated 600,000 deaths per year, mainly in developing countries (34). The development of a safe, effective rotavirus vaccine to provide protection 6093_05_p42-53 3/7/06 2:56 PM Page 42